The authors developed an in silico model that predicts the risk of drug-induced heart arrhythmias more accurately than animal studies.

They performed an ‘in silico drug trial’, testing 62 drugs and reference compounds at different concentrations on a control population of 1,213 simulated human ventricular cells.

Drug-induced changes in heart electrophysiology were measured in a user-friendly software, ‘Virtual Assay’, developed for this purpose and already used by four pharmaceutical partners. The computer model showed 89% accuracy in predicting the risk of drug-induced heart arrhythmias in humans, in comparison to up to 75% accuracy showed by data obtained from previously conducted animal studies.

Importantly the model also has the advantage that it represents the electrophysiological variability seen among patients. It outperforms other in silico approaches that average experimental data with a one-model-fits-all approach, and do not take inter-subject variability into account. This consideration of variability in simulations is crucial to capture differences in drug responses on a human population level and holds potential for identifying sub-populations at higher risk.

Read more in our news item, which includes videos from the research group at Oxford University who won the prize.

Highly Commended: Dr Christian Tiede, University of Leeds

The paper shows that it is possible to generate Affimers, ‘alternative binding proteins’ to numerous targets, validating the use of antibody alternative binding reagents in molecular and cellular research studies. They isolated Affimers against 12 different targets and compared them to equivalent antibodies across seven different case studies. The researchers showed that Affimers, because of their specificity and other factors, can be equal to, or better than, antibodies for various molecular and cell biology applications.

The ability to create Affimers in the laboratory without the use of animals means they could potentially replace (animal-made) antibodies in a number of common molecular and cellular applications.

The authors describe an alternative experimental design and analysis method – termed a ‘split-plot’ design – where animals are housed in mixed-strain groups. Female C57BL/6 (black), DBA/2 (brown) and BALB/c (white) mice were allocated to either enriched or standard treatments and screened for commonly measured and/ or welfare-relevant behavioural, physiological and haematological variables including corticosterone metabolite output and stereotypic behaviours. Results showed that living in mixed-strain trios did not reduce mouse welfare, or cause any change to strain-dependent or enrichment typical differences, validating it as an alternative study design.

The application of a ‘split-plot’ design meant fewer animals were required in each treatment group. The authors estimate, compared to a traditional design, this design can cut animal numbers required to achieve 80% power by more than half. Testing multiple strains increases the external validity of the experiment, thus increasing the generalisability of the results to other environmental contexts or populations.

2016

Prize winner: Dr Joanna Makowska, University of British Columbia

The authors provided evidence that natural behaviours such as burrowing and standing upright are important for the welfare of laboratory rats. They observed the behaviours of rats in two different cage types: standard laboratory caging and large, semi-naturalistic cages with a dedicated area for burrowing.

They found that throughout their lives rats burrow readily, even when tunnels are already present, re-organising the tunnels on an almost daily basis. When allowed the space in large cages, rats stand upright multiple times per day, even until relatively old, and climb often when young. In comparison, rats housed in standard laboratory caging are unable to carry out these behaviours, instead stretching laterally, suggesting that they are compensating for their inability to stretch in the natural upright position.

This work provides a scientific basis for a change in guidelines on laboratory rat housing, including increasing cage height and providing burrowing materials. A number of laboratories have already modified their rat housing to accommodate some of the features described in the paper.

The authors have developed the first 3D model of the human embryonic brain, using human induced pluripotent stem cells which were able to spontaneously self-organise into a structure that resembles the human brain with discrete, interdependent regions. This was achieved using adapted growth conditions, with specialised matrix support and improved access to nutrients through spinning. The paper also describes using skin cells from a patient with microcephaly to create organoids modelling the disease.

Suitability of animal models in studying neural development is limited, as they do not recapitulate the anatomical and functional complexity required to study human brain biology and disease. Developing brain organoids from human tissue is a revolutionary step towards reducing reliance on animals in studying neurological diseases and potentially in the development of new treatments.

The study, in collaboration with AstraZeneca, improves the technique of oral dosing in dogs. Following a framework of objective welfare assessments developed by Dr Hall, the authors demonstrate that a modified, refined protocol can minimise stress in dogs compared to the standard approach.

Most laboratory dogs in the UK are used for safety testing, and oral dosing is one of the most common procedures used during these tests. This paper shows that using seemingly small refinements (positive reinforcement training with food rewards, a signal for dosing, and covering the dosing tube in palatable paste during training) significantly reduces the negative welfare impact of the oral dosing on the dogs. The improved protocol allows researchers to dose dogs more quickly and efficiently, at no further cost. Dr Hall has been collaborating with dog facilities across the UK to maximise the impact of her work and share best practice.

Highly Commended: Dr Hayley Francies, Wellcome Trust Sanger Institute

The paper describes work on developing and characterising a biobank of colorectal cancer organoids obtained from patients’ biopsy tissue. The organoids were shown to accurately reflect the molecular features and diversity seen in the original tumours. More than 80 commonly used or experimental drugs for cancer were tested in the biobank.

Tumour-derived organoids have the potential to replace many studies involving patient-derived xenograft (PDX) mice, including for personalised medicine and drug development. Organoid production is cheaper and has a higher success rate than patient tumour sample engraftment rates in mice. This technology may fill the gap between cancer genetics and patient trials, complementing and in the long run replacing xenograft based drug studies, and allowing personalised therapy design.

The authors have built a computer model of cardiac electrophysiology that incorporates natural variability. Normally when using a computer model to test how a drug might affect the heart, the effect of the drug on the heart is compared to an average profile of electrophysiology. But this average profile is not really representative of the whole population, where natural variations in heart properties occur from person to person. This new approach has the potential to make computer models that are far more powerful and more predictive of human response, and therefore a more viable alternative to using animals in research. This is the first time that natural variability has successfully been considered in such a model, and the methodology could be applied to other diseases. The methodology has also been developed into a user-friendly software package called Virtual Assay, which is a major facilitator for industry uptake, without the need for specialist programming and modelling experience. The authors are already planning to use the same methodology to build computer models for understanding pain and diabetes.

Highly Commended: Dr Olivier Frey, ETH Zurich

Frey’s paper reports on a novel approach to culturing multi-cellular spheroids in vitro. The work is a significant advance in engineering, which brings together the hanging drop method and microfluidics to substantially expand the experimental options for culturing spheroids. Growing cells using a hanging drop approach, means that 3D cell spheroids can be grown without the restriction that may be imposed by a scaffold or a dish. Microfluidic systems allow precise liquid handling including continuous medium and waste exchange, and also allow test substances, such as candidate drugs, to be run through the culture. This is the first time these two approaches have been combined, and the result holds real promise to bolster the predictivity of in vitro research. The microfluidic hanging drop network has already shown that spheroids of cells representing different organs can be inter-connected in physiological order and are able to communicate with each other via metabolite transfer. This is exciting as this capability is one of the first steps towards creating a multi-organ body-on-a-chip model.

It is a requirement in animal safety testing of new medicines that the blood concentration of the medicine is measured. Historically, large volumes of blood were required to detect the concentration of the medicine. This meant that for rat studies, separate groups of rats were used solely for measuring the concentration while other groups of rats were used to assess the effects of the medicine on the animal. Advances in the way that blood is analysed mean that, with the right analysis equipment, very small “microsamples” of blood are now sufficient. Taking a microsample of blood from a rat is a quicker, less stressful procedure than taking a larger volume. This paper provides the evidence that taking repeat microsamples does not adversely affect adult rats and therefore does not interfere with the ability to interpret these safety studies. This means that information about the drug concentration and its affects can be obtained from the same animal, allowing a direct link between drug concentration and effect. This is a major scientific improvement. It also means that far fewer rats are required on these safety studies. As a consequence of this work, whenever the sensitive analytical method is available, AstraZeneca now use microsampling routinely in all rat safety tests.

Highly Commended: Drs Brianna Gaskill and Joseph Garner, Purdue University and Stanford University

Mice are commonly housed at temperatures (20–24°C) which humans find comfortable for working in the laboratory. Mice become cold stressed below 30˚C, which can compromise many aspects of physiology and welfare. However, the amount of nesting material required to meet a mouse’s thermal needs has until now been unknown. In this study the authors found out where mice wanted to spend their time, based on combinations of temperature and nesting material. They found that mice prefer temperatures between 26–29°C, but shift from preferring a warmer temperature to a nest when provided 6-10g of nesting material. These results suggest that laboratory mice should be provided with no less than 6g of nesting material in order to build fully formed nests, but 10g or more may be needed to eliminate thermal stress. These results have the potential to positively impact the welfare of millions of laboratory mice all over the world.

Dr Meritxell Huch from Cambridge University's Gurdon Institute wins the 2013 3Rs Prize for a Nature paper detailing work carried out at the Hubrecht Institute, The Netherlands, to develop a culture system that enables adult mouse stem cells to grow and expand into fully functioning three-dimensional liver tissue.

Growing hepatocytes (liver cells) in the laboratory has been attempted by liver biologists for many years, since it would reduce their reliance on using mice to study liver disease and would open up new opportunities in medical research and drug safety testing. Until now no laboratory has been successful in deciphering how to isolate and grow these cells.

Liver stem cells are typically found in a dormant state in the liver, only becoming active following injury to produce new liver cells and bile ducts. Dr Huch and colleagues located the specific type of stem cells responsible for this regeneration, which are recognised by a key surface protein (Lgr5+) that they share with similar stem cells in the intestine, stomach and hair follicles.

By isolating these cells and placing them in a culture medium with the right conditions, the researchers were able to grow small liver organoids, which survive and expand for over a year in a laboratory environment. When implanted back into mice with liver disease they continued to grow, ameliorating the disease and extending the survival of the mice.

Having further refined the process using cells from rats and dogs, Dr Huch is now moving onto testing it with human cells, which would not only be more relevant to research into human disease, but also translate to the development of a patient's own liver tissue for transplantation.

Commenting on the new method's potential to reduce animal use in liver research, Dr Huch said:

"Typically a study to investigate one potential drug compound to treat one form of liver disease would require up to 50 live animals per experiment, so testing 1000 compounds would need 50,000 mice. By using the liver culture system I developed, we can test 1000 compounds using cells that come from only one mouse, resulting in a significant reduction in animal use.

Growing functioning liver cells in culture has been the Holy Grail for liver biologists for many years, so a limitless supply of hepatocytes could have a huge 3Rs impact both on basic research to understand liver disease and for the screening and safety testing of pharmaceuticals.

Professor Ingber's research, published in Science Translational Medicine, describes an innovative 'lung-on-a-chip' microdevice that can accurately replicate conditions in a diseased human lung, offering a viable alternative to using animals in preclinical drug testing.

The microdevice contains hollow channels lined with living human cells, mimicking both the interface between tissues and the unique physical environment seen in whole living organs. Crystal clear and flexible, it is approximately the size of a USB memory stick.

Applying a vacuum to part of the microdevice allows it to 'breathe', recreating the way in which our tissues physically expand and contract during respiration. In testing it was able to successfully replicate the conditions seen in pulmonary oedema (fluid accumulation in the lungs), and predict results of a new drug for this life-threatening condition, which showed benefit in animal studies.

In addition, the microdevice has allowed the researchers to carry out real-time high resolution imaging on the cells and make accurate measurements of fluid flow and blood clot formation, which are not easily available in an animal model.

Professor Ingber said: "This is precisely the kind of progress that regulatory government agencies, such as the Food and Drug Administration (FDA) in the US, and pharmaceutical companies need to see in order to seriously consider an alternative approach to animal models."

In their paper the researchers describe how the next step is to apply the technology to other human organs with the goal of one day being able to use it as part of an automated system to test many drugs. While it is not expected to offer an immediate replacement for animal studies, further development and applications of the technology could allow for a more gradual replacement of animal models of human disease.

How the lung-on-a-chip works:

Inside the microdevice are two parallel, sub-millimeter sized, hollow channels which are separated by a thin, flexible, porous membrane. This membrane is coated with matrix proteins that normally hold cells together in human tissues.

One side of this membrane is lined with living human cells isolated from the air sac of a lung, and air is allowed to permeate into the channel to recreate the environment seen in a lung. The other side contains human lung capillary blood cells with a blood-like solution flowing over their surfaces.

A vacuum applied to side chambers alongside the channels recreates the way our tissues physically expand and retract when we breathe.

Recreating these conditions has been an important step to develop new insights into human lung disease that are difficult to achieve in with animal studies, such as the ability to carry out high-resolution imaging on the cells themselves, observing blood clot formation and fluid flow.

Scottish-based researcher Professor Susan Barnett was commended for research developing an in vitro model of spinal cord injury using rat embryonic spinal cord cells. This has enabled the laboratory to test the combination of drugs being studied using cells from one animal only, representing a 97% reduction had an established methodology been used. This method is being further developed for testing therapeutics more widely.

London-based Professor Gareth Sanger was commended for research demonstrating the benefit of using human - rather than animal - gastrointestinal tissues for drug testing, which are obtained as part of normal surgical procedures.

US researcher Professor Shuichi Takayama was commended for developing a 3D cell culture to test anti-cancer drugs, which proved to be more representative of clinical responses than standard 2D 'flat' cell cultures, demonstrating the potential for this method to replace and reduce the use of animals in pharmaceutical testing.

Dr Vallier's paper looks at the use of artificial liver cells to model inherited metabolic disorders of the liver has the potential to reduce the number of animals used in this type of research.

The artificial cells, known as human induced pluripotent stem cells (hIPSCs), offer possibilities to regenerate damaged tissues and organs, and it is their potential to reduce the number of animals used for screening potential drug treatments that led to Dr Vallier receiving the 3Rs Prize in 2011.

Human liver cells (hepatocytes) cannot be grown in the laboratory and differences between rodents and humans mean that it is rarely possible to recreate the human disease completely in mice or rats or to use cultures of rat or mouse liver cells. Dr Vallier's team took skin cells (dermal fibroblasts) from seven patients with a variety of inherited liver diseases and three healthy individuals (the controls). They then reprogrammed cells from the skin samples back into stem cells. These stem cells were then used to generate liver cells which mimicked a broad range of liver diseases and to create 'healthy' liver cells from the control group.

Ludovic Vallier's innovative study describes the development and validation of a method to produce cells similar to those in a human liver. Such cells could replace animals for some types of early drug testing and could also help us to predict adverse clinical reactions. Using these cells for drug testing could be transformative. Ludovic and his colleagues have well illustrated how addressing the 3Rs converges with improving the quality of science.

Highly Commended: Dr Anna Williams, MRC Centre for Regenerative Medicine at the University of Edinburgh

In multiple sclerosis (MS), immune cells enter the brain and cause inflammation and demyelination, where the protective covering of myelin around nerves is damaged. The brain can repair this damage by a process called remyelination, but this is not very efficient and frequently fails. Researchers have used rats and mice to try reproduce demyelination and to test for possible medicines to help promote remyelination. These experiments use large numbers of animals.

Dr Williams discovered that slices of brain taken from very young mice and grown in a dish, retain the three-dimensional structure and normal cells of the brain and can be used to test the effectiveness of medicines that might improve remyelination. The researchers also automated the ways in which they measured the remyelination, such that analysis now takes seconds rather than hours.

Highly Commended: Dr Stephen Pettit, Wellcome Trust Sanger Institute

Research conducted by Dr Pettitt and colleagues at the Wellcome Trust Sanger Insitute has provided a new way of generating GM mice which avoids using two different strains and therefore reduces the number of animals used.

Scientists use genetically modified (GM) mice to study a range of diseases. But producing GM mice can be a lengthy process involving large numbers of animals. For technical reasons scientists often use a particular strain of mice for the early parts of the process. However, they then need to breed the mice with another strain to give animals with the desired genetic background for their experiments.

The 'Black 6' strain of mouse (so-called because of its coat colour) is preferred for many experiments. However, for years GM mice could only be made efficiently in a different strain, '129', from which it was easy to derive embryonic stem cells. This meant the GM 129 mice had to be bred with Black 6 mice for several generations (backcrossing) before the mutation could be analysed on a Black 6 genetic background.

This technique has the potential to reduce the numbers of mice used by hundreds per project. The cells are already in use in an international project to investigate all 20,000 mouse genes, and this can now be done with increased efficiency and fewer animals.

Dr Pettitt's work consisted of deriving embryonic stem cells the starting point for making a GM mouse directly from Black 6 mice, thus removing the need for backcrossing. Importantly, the cells were modified so that they could be easily seen by a difference in coat colour in the mice. This means that mice with the highest proportion of GM cells can be selected for breeding ensuring that only those animals likely to transmit the genetic modification to their offspring are used.

2010

Prize winner: Professor Jane Hurst, University of Liverpool

Professor Jane Hurst's research has shown that a new way of handling laboratory mice can improve their welfare and the quality of the science they are used for.

Laboratory mice are usually picked up by their tails. Professor Hurst's study proves this method of handling causes high levels of anxiety and stress which can influence the outcome of experiments. By simply catching the mice using a plastic tunnel or cupped hands anxiety can be greatly reduced. This small change can be easily applied and has the potential to make a big difference to the welfare of every mouse used for research.

Dr Jenny Nichols developed an optimised culture medium for growing mouse embryonic stem (ES) cells and utilised it to derive ES cells from non-obese diabetic (NOD) mice for the first time. This could dramatically reduce the number of animals used to study the genetic basis of type 1 diabetes and also has the potential to do the same for mouse models of other diseases.

ES cells are derived from early embryos and can be grown indefinitely in culture. They are a powerful tool in biomedical research because of their 'pluripotency', which means they can be transformed into all the cell types in the body, making them widely used in in vitro experiments and to generate disease models in mice.

Previously, understanding which genes play a role in type 1 diabetes involved breeding NOD mice with strains which had the gene of interest modified. This lengthy process required at least ten generations of breeding, involving many hundreds of animals, before the mice had a suitable genetic background for conducting the experiment.

The derivation of ES cells from the NOD mouse allows its genes to be directly manipulated to study type 1 diabetes without many generations of backcrossing, dramatically reducing the number of mice required per experiment. The NOD ES cells are now freely available to the research community, potentially reducing the number of mice used in tpe 1 diabetes research worldwide.

The new medium, which contains a novel mixture of cell growth factors and inhibitors but no animal products, has also allowed the derivation of ES cells from every strain of mouse tested so far with extremely high efficiency. Dr Nichols' laboratory has made this technology available so that it can be applied to other mouse models of disease where deriving ES cells has previously proven impossible.

Dr Martin, and his colleague Mr Thomas Johnson, work investigates the potential of stem cells to protect vulnerable nerve cells in the injured retina. Their aim is to develop new treatments for glaucoma, the leading cause of irreversible blindness worldwide, and other eye diseases. Dr Martin and Mr Johnson pioneered a new method for retinal tissue culture that replaces the need for experiments on live animals.

They have shown that the cultured eye tissue remains healthy, maintains its layered architecture, and retains the ability to make new proteins. The tissue also responds to stem cell transplantation in a similar way to the eyes of living animals.

As well as replacing the use of live animals, the new method has brought about an eight-fold reduction in the number of animals used, because eight sections of tissue can be obtained from one rat.

In her research, Dr Gower worked on schistosomes, the worm-like parasites that cause bilharzia, a tropical disease affecting an estimated 200 million people worldwide. The disease can be seriously debilitating, causing long term liver and intestinal damage, and can sometimes be fatal. Dr Gower's prize has been awarded for a new application of DNA fingerprinting which replaces the need for using animals in this research area and improves the value and accuracy of her results.

Dr Gower has taken advantage of recent advances in how DNA can be stored at room temperature and characterised from minute samples in order to collect parasite DNA samples directly from infected people in areas where the disease is endemic. Previously, there was a need to grow the parasites in the laboratory for study by collecting worm eggs from human faeces and using them to infect snails and then rodents.

In their research, Professor Fairlamb and Dr Wyllie infect hamsters with the parasite Leishmania donovani which causes visceral leishmaniasis. By using a different route of infection, the duration and severity of the disease in the hamster was reduced without compromising the quality of the scientific outcome.

Professor Fairlamb compared the commonly-used intracardial route of infection with the intraperitoneal route. The results showed that the intraperitoneal route is a simpler, safer and effective method of inoculating the hamsters.

In her research, Dr Wiles infects mice with bacteria from the same family as E. coli to study the paths of infection. Traditionally, every mouse has been infected by putting a tube down its throat to deliver the bacteria to the stomach - a process called gavage.

Dr Wiles tried infecting only one mouse in this way, then putting it in a cage with uninfected mice and letting nature take its course.

The results showed higher infection rates than the traditional technique. But more importantly, the research was refined so that far fewer animals were subjected to gavage, and the new approach also reduced the total number of animals used by improving the reliability of infection.